T2511
DNA from up to 200 l of Intestinal mucus samples was extracted using a High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland), including a lysozyme lysis step for optimized DNA extraction (Piazzon et al., 2019). For intestinal microbiota analysis, the V3-V4 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform (2300paired-end run) at the Genomics Unit from the Madrid Science Park Foundation (FPCM) as described elsewhere (Piazzon et al., 2019). Three samples, one per group, failed amplification and were removed for further analysis. FASTQ forward and reverse reads were quality-filtered and pre-processed using FastQC (Andrews et al., 2010) and Prinseq v0.20.4 (Schmieder and Edwards, 2011). Terminal N bases in both ends were trimmed, and sequences with >5% N bases, <150 bp long, Phred quality score <28 in both ends, or Phred average score <26 were discarded. Clean forward and reverse reads were merged with fastq-join (Aronesty, 2013).